Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hba, and Tyr280 and Trp284 in Hp beta. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb beta Trp37 and Hp beta Phe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.

Automated summary

Structural mass spectrometry methods, particularly FPOP, are widely used for studying protein structure and dynamics. In this study, FPOP analysis of the Hb-Hp complex revealed specific residues affected by the complex formation and provided insights into the interacting amino acids. The data, available via ProteomeXchange, can be further utilized for investigating protein-protein interactions.